Mechanisms of the 3'-5' deoxyribonucleases

3-5脱氧核糖核酸酶的机制

• 批准号: 6941648
• 负责人: FRED W PERRINO
• 金额: $ 28.7万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941648
• 关键词: Drosophilidae; RNA interference; X ray crystallography; active sites; binding sites; biochemistry; catalyst; dimer; endodeoxyribonuclease; enzyme activity; enzyme substrate complex; genetic models; liquid chromatography mass spectrometry; model design /development; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; recombinant proteins; site directed mutagenesis

DESCRIPTION (provided by applicant): The studies proposed in this new grant application will examine structure, mechanism, and function of the 3'-->5' deoxyribonucleases encoded by the TREX (Three prime Repair EXonuclease) genes. The 3'-->5' deoxyribonucleases are essential enzymes in DNA metabolism that catalyze excision of nucleotides from the 3' ends of DNA to prepare these 3' termini for subsequent steps during DNA replication, repair, and recombination. The 3' deoxyribonucleases excise mismatched, modified, fragmented, or normal nucleotides from DNA 3' termini, and the actions of these enzymes are critical in many DNA metabolic pathways that maintain genomic integrity in all organisms. While the existence of 3' deoxyribonuclease activities in eucaryotes has been recognized for more than thirty years, only recently have some of the genes encoding these deoxyribonucleases been identified. The TREX genes identified in this laboratory are present in metazoans and encode proteins that are members of a larger nuclease family that includes both deoxy- and ribo-exonucleases. The deoxyribonucleases in this family include large multiple-domain proteins as well as smaller single-domain proteins. There is currently insufficient information about the 3'-->5' deoxyribonucleases functioning in human cells to understand the mechanisms by which these proteins recognize and excise 3' nucleotides making it difficult to dicern the molecular pathways in which these proteins function. The goal of experiments in this proposal is to seek a better understanding of the biochemistry of the TREX 3'-->5' exonucleases and to establish a genetic system to address TREX protein function in vivo. As a first step, we have cloned the metazoan TREX genes and established an expression system to produce these proteins for biochemical studies. Aim 1 of this project is to generate the recombinant TREX proteins and site-directed mutants in sufficient quantities for mechanistic and structural studies. In aim 2, mechanistic studies of the enzymes will be used in conjunction with NMR studies to quantify enzyme-substrate interactions and to determine the nature of substrate recognition and specificity in the TREX proteins. Experiments in aim 3 focus on X-ray structural studies of the TREX proteins. These structural studies will provide insights into the catalytic site, dimer interface, and nucleic acid binding surfaces. Experiments in aim 4 will address issues of function by identifying TREX protein binding partners and by establishing a genetic system in Drosophila. Identification of the TREX genes encoding these 3'-->5' deoxyribonucleases has made possible these mechanistic studies that will provide new insights into the physiological function of these enzymes.

描述（由申请人提供）： 这项新拨款申请中提出的研究将检查 TREX（三素修复核酸外切酶）基因编码的 3'-->5' 脱氧核糖核酸酶的结构、机制和功能。 3'-->5' 脱氧核糖核酸酶是 DNA 代谢中必需的酶，可催化 DNA 3' 末端的核苷酸切除，为 DNA 复制、修复和重组过程中的后续步骤准备这些 3' 末端。 3' 脱氧核糖核酸酶从 DNA 3' 末端切除不匹配、修饰、片段化或正常的核苷酸，这些酶的作用在许多维持所有生物体基因组完整性的 DNA 代谢途径中至关重要。尽管三十多年来人们已经认识到真核生物中存在 3' 脱氧核糖核酸酶活性，但直到最近才鉴定出一些编码这些脱氧核糖核酸酶的基因。本实验室鉴定的 TREX 基因存在于后生动物中，编码的蛋白质是更大的核酸酶家族的成员，该家族包括脱氧核酸外切酶和核糖核酸外切酶。该家族中的脱氧核糖核酸酶包括大型多结构域蛋白质以及较小的单结构域蛋白质。目前关于 3'->5' 脱氧核糖核酸酶在人类细胞中发挥作用的信息不足，无法了解这些蛋白质识别和切除 3' 核苷酸的机制，因此很难确定这些蛋白质发挥作用的分子途径。本提案中的实验目标是更好地了解 TREX 3'-->5' 核酸外切酶的生物化学，并建立一个遗传系统来解决体内 TREX 蛋白功能。第一步，我们克隆了后生动物 TREX 基因并建立了表达系统来生产这些蛋白质用于生化研究。该项目的目标 1 是生成足够数量的重组 TREX 蛋白和定点突变体，用于机制和结构研究。在目标 2 中，酶的机理研究将与 NMR 研究结合使用，以量化酶-底物相互作用并确定 TREX 蛋白中底物识别和特异性的性质。目标 3 中的实验侧重于 TREX 蛋白的 X 射线结构研究。这些结构研究将提供对催化位点、二聚体界面和核酸结合表面的深入了解。目标 4 中的实验将通过识别 TREX 蛋白结合伴侣并在果蝇中建立遗传系统来解决功能问题。编码这些 3'-->5' 脱氧核糖核酸酶的 TREX 基因的鉴定使这些机制研究成为可能，这些研究将为这些酶的生理功能提供新的见解。